RESUMO
For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.
Assuntos
Metadados , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Aplicativos Móveis , Linguagens de Programação , Software , Animais , Linhagem Celular , Biologia Computacional/métodos , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Reconhecimento Automatizado de Padrão , Controle de Qualidade , Reprodutibilidade dos Testes , Interface Usuário-Computador , Fluxo de TrabalhoRESUMO
DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Animais , Apoptose/genética , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/biossíntese , Regulação da Expressão Gênica , Neuropatias Hereditárias Sensoriais e Autônomas/patologia , Heterocromatina/genética , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Neurônios/metabolismo , Neurônios/patologia , Domínios Proteicos/genética , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Ubiquitina-Proteína LigasesAssuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Processamento de Imagem Assistida por Computador/normas , Informática Médica/estatística & dados numéricos , Metadados/estatística & dados numéricos , Microscopia/métodos , Software , Humanos , Processamento de Imagem Assistida por Computador/métodosRESUMO
Spontaneous network activity is a highly stereotyped early feature of developing circuits throughout the nervous system, including in the spinal cord. Spinal locomotor circuits produce a series of behaviors during development before locomotion that reflect the continual integration of spinal neurons into a functional network, but how the circuitry is reconfigured is not understood. The first behavior of the zebrafish embryo (spontaneous coiling) is mediated by an electrical circuit that subsequently generates mature locomotion (swimming) as chemical neurotransmission develops. We describe here a new spontaneous behavior, double coiling, that consists of two alternating contractions of the tail in rapid succession. Double coiling was glutamate-dependent and required descending hindbrain excitation, similar to but preceding swimming, making it a discrete intermediary developmental behavior. At the cellular level, motoneurons had a distinctive glutamate-dependent activity pattern that correlated with double coiling. Two glutamatergic interneurons, CoPAs and CiDs, had different activity profiles during this novel behavior. CoPA neurons failed to show changes in activity patterns during the period in which double coiling appears, whereas CiD neurons developed a glutamate-dependent activity pattern that correlated with double coiling and they innervated motoneurons at that time. Additionally, double coils were modified after pharmacological reduction of glycinergic neurotransmission such that embryos produced three or more rapidly alternating coils. We propose that double coiling behavior represents an important transition of the motor network from an electrically coupled spinal cord circuit that produces simple periodic coils to a spinal network driven by descending chemical neurotransmission, which generates more complex behaviors.
Assuntos
Atividade Motora/fisiologia , Neurônios Motores/fisiologia , Rede Nervosa/fisiologia , Medula Espinal/citologia , Sinapses/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Estimulação Elétrica , Embrião não Mamífero , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Vias Neurais/embriologia , Rombencéfalo/fisiologia , Medula Espinal/embriologia , Sinapses/classificação , Sinapses/efeitos dos fármacos , Fatores de Transcrição/genética , Valina/análogos & derivados , Valina/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to scale coordinately with mitotic spindles, but the underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle, and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be reset by cytoplasmic factors from earlier developmental stages. In vitro, increasing nuclear-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin α scales mitotic chromosomes to cell surface area/volume ratio (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and Hi-C data suggest that mitotic chromosomes shrink during embryogenesis through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter chromosome axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.
Assuntos
Núcleo Celular , Cromossomos , Animais , Xenopus laevis/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Fuso Acromático/metabolismo , Tamanho Celular , MitoseRESUMO
Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.
RESUMO
Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.
Commercially produced antibodies are essential research tools. Investigators at universities and pharmaceutical companies use them to study human proteins, which carry out all the functions of the cells. Scientists usually buy antibodies from commercial manufacturers who produce more than 6 million antibody products altogether. Yet many commercial antibodies do not work as advertised. They do not recognize their intended protein target or may flag untargeted proteins. Both can skew research results and make it challenging to reproduce scientific studies, which is vital to scientific integrity. Using ineffective commercial antibodies likely wastes $1 billion in research funding each year. Large-scale validation of commercial antibodies by an independent third party could reduce the waste and misinformation associated with using ineffective commercial antibodies. Previous research testing an antibody validation pipeline showed that a commercial antibody widely used in studies to detect a protein involved in amyotrophic lateral sclerosis did not work. Meanwhile, the best-performing commercial antibodies were not used in research. Testing commercial antibodies and making the resulting data available would help scientists identify the best study tools and improve research reliability. Ayoubi et al. collaborated with antibody manufacturers and organizations that produce genetic knock-out cell lines to develop a system validating the effectiveness of commercial antibodies. In the experiments, Ayoubi et al. tested 614 commercial antibodies intended to detect 65 proteins involved in neurologic diseases. An effective antibody was available for about two thirds of the 65 proteins. Yet, hundreds of the antibodies, including many used widely in studies, were ineffective. Manufacturers removed some underperforming antibodies from the market or altered their recommended uses based on these data. Ayoubi et al. shared the resulting data on Zenodo, a publicly available preprint database. The experiments suggest that 20-30% of protein studies use ineffective antibodies, indicating a substantial need for independent assessment of commercial antibodies. Ayoubi et al. demonstrated their side-by-side antibody comparison methods were an effective and efficient way of validating commercial antibodies. Using this approach to test commercial antibodies against all human proteins would cost about $50 million. But it could save much of the $1 billion wasted each year on research involving ineffective antibodies. Independent validation of commercial antibodies could also reduce wasted efforts by scientists using ineffective antibodies and improve the reliability of research results. It would also enable faster, more reliable research that may help scientists understand diseases and develop new therapies to improve patient's lives.
Assuntos
Anticorpos , Proteoma , Humanos , Anticorpos/químicaRESUMO
The molecular and physiological basis of the touch-unresponsive zebrafish mutant fakir has remained elusive. Here we report that the fakir phenotype is caused by a missense mutation in the gene encoding voltage-gated calcium channel 2.1b (CACNA1Ab). Injection of RNA encoding wild-type CaV2.1 restores touch responsiveness in fakir mutants, whereas knockdown of CACNA1Ab via morpholino oligonucleotides recapitulates the fakir mutant phenotype. Fakir mutants display normal current-evoked synaptic communication at the neuromuscular junction but have attenuated touch-evoked activation of motor neurons. NMDA-evoked fictive swimming is not affected by the loss of CaV2.1b, suggesting that this channel is not required for motor pattern generation. These results, coupled with the expression of CACNA1Ab by sensory neurons, suggest that CaV2.1b channel activity is necessary for touch-evoked activation of the locomotor network in zebrafish.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Ativação do Canal Iônico/genética , Tato/genética , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Vias Aferentes/fisiologia , Animais , Animais Geneticamente Modificados , Bungarotoxinas/metabolismo , Canais de Cálcio Tipo N/genética , Curare/farmacologia , Relação Dose-Resposta a Droga , Embrião não Mamífero , Reação de Fuga/efeitos dos fármacos , Reação de Fuga/fisiologia , Potenciais Evocados/genética , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Leucina/genética , Locomoção/efeitos dos fármacos , Locomoção/genética , Modelos Moleculares , Morfolinas/farmacologia , Atividade Motora/genética , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Mutagênese Sítio-Dirigida/métodos , Mutação/genética , Mutação de Sentido Incorreto/genética , Rede Nervosa/fisiologia , Antagonistas Nicotínicos/farmacologia , Medula Espinal/citologia , Medula Espinal/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Tato/fisiologia , Valina/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The process by which light touch in vertebrates is transformed into an electrical response in cutaneous mechanosensitive neurons is a largely unresolved question. To address this question we undertook a forward genetic screen in zebrafish (Danio rerio) to identify mutants exhibiting abnormal touch-evoked behaviors, despite the presence of sensory neurons and peripheral neurites. One family, subsequently named touché, was found to harbor a recessive mutation which produced offspring that were unresponsive to light touch, but responded to a variety of other sensory stimuli. The optogenetic activation of motor behaviors by touché mutant sensory neurons expressing channelrhodopsin-2 suggested that the synaptic output of sensory neurons was intact, consistent with a defect in sensory neuron activation. To explore sensory neuron activation we developed an in vivo preparation permitting the precise placement of a combined electrical and tactile stimulating probe upon eGFP-positive peripheral neurites. In wild-type larva electrical and tactile stimulation of peripheral neurites produced action potentials detectable within the cell body. In a subset of these sensory neurons an underlying generator potential could be observed in response to subthreshold tactile stimuli. A closer examination revealed that the amplitude of the generator potential was proportional to the stimulus amplitude. When assayed touché mutant sensory neurons also responded to electrical stimulation of peripheral neurites similar to wild-type larvae, however tactile stimulation of these neurites failed to uncover a subset of sensory neurons possessing generator potentials. These findings suggest that touché is required for generator potentials, and that cutaneous mechanoreceptors with generator potentials are necessary for responsiveness to light touch in zebrafish.
Assuntos
Potenciais Somatossensoriais Evocados/fisiologia , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais/fisiologia , Tato/fisiologia , Proteínas de Peixe-Zebra/genética , Animais , Eletrofisiologia , Rede Nervosa/fisiologia , Neurônios Aferentes/fisiologia , Estimulação Física , Peixe-Zebra/genéticaRESUMO
Liquid-liquid phase separation (LLPS) mediated formation of membraneless organelles has been proposed to coordinate biological processes in space and time. Previously, the formation of phase-separated droplets was described as a unique property of HP1α. Here, we demonstrate that the positive net charge of the intrinsically disordered hinge region (IDR-H) of HP1 proteins is critical for phase separation and that the exchange of four acidic amino acids is sufficient to confer LLPS properties to HP1ß. Surprisingly, the addition of mono-nucleosomes promoted H3K9me3-dependent LLPS of HP1ß which could be specifically disrupted with methylated but not acetylated H3K9 peptides. HP1ß mutants defective in H3K9me3 binding were less efficient in phase separationin vitro and failed to accumulate at heterochromatin in vivo. We propose that multivalent interactions of HP1ß with H3K9me3-modified nucleosomes via its chromodomain and dimerization via its chromoshadow domain enable phase separation and contribute to the formation of heterochromatin compartments in vivo.
Assuntos
Proteínas Cromossômicas não Histona , Histonas , Condensados Biomoleculares , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina , Histonas/genética , Histonas/metabolismo , MetilaçãoRESUMO
Although many components of the cell division machinery in bacteria have been identified1,2, the mechanisms by which they work together to divide the cell remain poorly understood. Key among these components is the tubulin FtsZ, which forms a Z ring at the midcell. FtsZ recruits the other cell division proteins, collectively called the divisome, and the Z ring constricts as the cell divides. We applied live-cell single-molecule imaging to describe the dynamics of the divisome in detail, and to evaluate the individual roles of FtsZ-binding proteins (ZBPs), specifically FtsA and the ZBPs EzrA, SepF and ZapA, in cytokinesis. We show that the divisome comprises two subcomplexes that move differently: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a moving complex that includes cell wall synthases. Our imaging analyses reveal that ZBPs bundle FtsZ filaments together and condense them into Z rings, and that this condensation is necessary for cytokinesis.
Assuntos
Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citocinese , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Ligação Proteica , Imagem Individual de MoléculaRESUMO
Mutations in the human PTEN-induced kinase 1 (PINK1) gene are linked to recessive familial Parkinson's disease. Animal models of altered PINK1 function vary greatly in their phenotypic characteristics. Drosophila pink1 mutants exhibit mild dopaminergic neuron degeneration and locomotion defects. Such defects are not observed in mice with targeted null mutations in pink1, although these mice exhibit impaired dopamine release and synaptic plasticity. Here, we report that in zebrafish, morpholino-mediated knockdown of pink1 function did not cause large alterations in the number of dopaminergic neurons in the ventral diencephalon. However, the patterning of these neurons and their projections are perturbed. This is accompanied by locomotor dysfunction, notably impaired response to tactile stimuli and reduced swimming behaviour. All these defects can be rescued by expression of an exogenous pink1 that is not a target of the morpholinos used. These results indicate that normal PINK1 function during development is necessary for the proper positioning of populations of dopaminergic neurons and for the establishment of neuronal circuits in which they are implicated.
Assuntos
Diencéfalo/crescimento & desenvolvimento , Proteínas Quinases/genética , Natação/fisiologia , Percepção do Tato/fisiologia , Peixe-Zebra , Sequência de Aminoácidos , Animais , Diencéfalo/anatomia & histologia , Diencéfalo/efeitos dos fármacos , Diencéfalo/metabolismo , Dopamina/metabolismo , Larva/efeitos dos fármacos , Larva/fisiologia , Dados de Sequência Molecular , Neurônios/metabolismo , Neurônios/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Proteínas Quinases/metabolismoRESUMO
Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.
Assuntos
Diferenciação Celular , Citosina/metabolismo , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas/genética , Animais , Sistemas CRISPR-Cas , Cromatografia Líquida de Alta Pressão , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Epigênese Genética , Camundongos , Camundongos Knockout , Proteoma , Proteômica , Proteínas Proto-Oncogênicas/metabolismo , Espectrometria de Massas em TandemRESUMO
Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona , Desmetilação do DNA , Mamíferos/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Evolução Biológica , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , DNA Polimerase Dirigida por DNA/metabolismo , Epigenômica , Evolução Molecular , Regulação da Expressão Gênica , Genes Reguladores , Células Germinativas/metabolismo , Camundongos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Assembling composite DNA modules from custom DNA parts has become routine due to recent technological breakthroughs such as Golden Gate modular cloning. Using Golden Gate, one can efficiently assemble custom transcription units and piece units together to generate higher-order assemblies. Although Golden Gate cloning systems have been developed to assemble DNA plasmids required for experimental work in model species, they are not typically applicable to organisms from other kingdoms. Consequently, a typical molecular biology laboratory working across kingdoms must use multiple cloning strategies to assemble DNA constructs for experimental assays. To simplify the DNA assembly process, we developed a multi-kingdom (MK) Golden Gate assembly platform for experimental work in species from the kingdoms Fungi, Eubacteria, Protista, Plantae, and Animalia. Plasmid backbone and part overhangs are consistent across the platform, saving both time and resources in the laboratory. We demonstrate the functionality of the system by performing a variety of experiments across kingdoms including genome editing, fluorescence microscopy, and protein interaction assays. The versatile MK system therefore streamlines the assembly of modular DNA constructs for biological assays across a range of model organisms.
Assuntos
Clonagem Molecular/métodos , Edição de Genes , Proteínas Recombinantes/genética , Animais , Bactérias/genética , Feminino , Humanos , Oócitos/fisiologia , Organismos Geneticamente Modificados , Plantas/genética , Plasmídeos/genética , Proteínas/análise , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Transcrição Gênica , Transgenes , Trypanosoma/genética , Xenopus laevis , Leveduras/genéticaRESUMO
Rod-shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: the Rod complex moves around the cell circumference, whereas class A penicillin-binding proteins (aPBPs) do not. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each affects the growth and width of Bacillus subtilis as well as the mechanical anisotropy and orientation of material within their sacculi. Rod width is not determined by MreB, rather it depends on the balance between the systems: the Rod complex reduces diameter, whereas aPBPs increase it. Increased Rod-complex activity correlates with an increased density of directional MreB filaments and a greater fraction of directional PBP2a enzymes. This increased circumferential synthesis increases the relative quantity of oriented material within the sacculi, making them more resistant to stretching across their width, thereby reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems builds and maintains rods of different widths. Escherichia coli Rod mutants also show the same correlation between width and directional MreB filament density, suggesting this model may be generalizable to bacteria that elongate via the Rod complex.
Assuntos
Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Parede Celular/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/metabolismoRESUMO
For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.
Assuntos
Microscopia/métodos , Animais , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia/classificação , Microscopia/instrumentação , Microscopia/normas , Óptica e FotônicaRESUMO
Super-resolution microscopy is a very powerful tool to investigate fine cellular structures and molecular arrangements in biological systems. For instance, stimulated emission depletion (STED) microscopy has been successfully used in recent years to investigate the arrangement and colocalization of different protein species in cells in culture and on the surface of specimens. However, because of its extreme sensitivity to light scattering, super-resolution imaging deep inside tissues remains a challenge. Here, we describe the preparation of thin slices from the fruit fly (Drosophila melanogaster) brain, subsequent immunolabeling and imaging with STED microscopy. This protocol allowed us to image small dendritic branches from neurons located deep in the fly brain with improved resolution compared with conventional light microscopy.
Assuntos
Encéfalo/metabolismo , Drosophila/metabolismo , Microscopia de Fluorescência/métodos , Animais , Crioultramicrotomia/métodos , Espinhas Dendríticas/metabolismo , Imunofluorescência , Imagem Molecular , Neurônios/metabolismoRESUMO
Ubiquitination is a multifunctional posttranslational modification controlling the activity, subcellular localization and stability of proteins. The E3 ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) is an essential epigenetic factor that recognizes repressive histone marks as well as hemi-methylated DNA and recruits DNA methyltransferase 1. To explore enzymatic functions of UHRF1 beyond epigenetic regulation, we conducted a comprehensive screen in mouse embryonic stem cells to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2. We found differentially ubiquitinated peptides associated with a variety of biological processes such as transcriptional regulation and DNA damage response. Most prominently, we identified PCNA-associated factor 15 (PAF15; also known as Pclaf, Ns5atp9, KIAA0101 and OEATC-1) as a specific ubiquitination target of UHRF1. Although the function of PAF15 ubiquitination in translesion DNA synthesis is well characterized, the respective E3 ligase had been unknown. We could show that UHRF1 ubiquitinates PAF15 at Lys 15 and Lys 24 and promotes its binding to PCNA during late S-phase. In summary, we identified novel ubiquitination targets that link UHRF1 to transcriptional regulation and DNA damage response.